Biological oxidations catalyzed by Heme Protein Reductases represent one of the most important and ubiquitous reactions in biochemistry. Paramount players in the metabolic transformations operating in all life forms, the cytochromes P450 are critical components of animal, plant, insect, bacterial, archaeal and invertebrate physiology. They are responsible for the oxygenation of a plethora of compounds, including cholesterol and steroid structures, long chain alkanes, aromatics, fatty acids, drugs, insecticides, pesticides, critical signaling molecules and carcinogen precursors. Through the research supported by this continuing grant, we seek to reveal the detailed mechanisms that are common to all P450s with major effort on those cytochromes P450 that play central roles in human pathways, including steroid hormone biosynthesis and drug metabolism. Specific Aims for the next funding period are: (I) To reveal Nature's means for catalyzing human steroid hormone biosynthesis, focusing on the carbon- carbon bond scission reactions catalyzed by three human P450 isozymes. These include the initial committed step in steroid biosynthesis that forms pregnenolone from cholesterol, the critical step in androgen formation via the cleavage of the C17-C20 bond to generate the characteristic keto group at the apex of the D-ring in androgens and the reaction catalyzed by the important drug target, aromatase, wherein an aromatic A-ring is formed concomitantly with cleavage of the C10-C19 bond. (II) To fully characterize the chemical mechanisms of P450s involved in human drug metabolism, the detailed enzymology behind clinically important drug- drug interactions, the identity of heme-oxygen intermediate states in the catalytic cycle and the fate of reducing equivalents in critical uncoupling reactions that produce reactive oxygen intermediates. (III) Continue to utilize innovative biochemical and biophysical means to understand the global mechanisms of heme protein reductase catalysis, thus precisely defining the structure and dynamics of key intermediates in the control of electron and proton delivery. Through this integrated approach, using a wide variety of chemical and biophysical methodologies, we believe that the next funding period will realize a deep understanding of mixed function oxidase catalysis, provide important insights into normal functioning as well the lesions associated with human disease and thus dramatically aiding intervention through novel therapeutic approaches. PUBLIC HEALTH RELEVANCE: The cytochromes P450 play critical roles in human metabolism. Our focused research on P450s at the control points of steroid biosynthesis in man involves P450 CYP19 (aromatase), which is the key target of the latest generation of drugs used to fight breast cancer. P450 CYP17 is a promising prostate cancer target, and P450 CYP11A1 is the initial step to make all steroid hormones. P450 CYP3A4 in human liver metabolizes a majority of the most commonly prescribed drugs, and is the site for clinical manifestation of many deleterious drug-drug interactions. Detailed mechanistic understanding of these systems will provide insights that enable the development of safer and more effective therapeutic interventions.